With the emergence of bacterial resistance, identification of new diseases, and the need for new therapeutics with different efficacies, our ability to design new drugs is becoming a more urgent priority. Natural products are often useful as therapeutics for humans, though problems such as side effects and production difficulties can preclude their successful development. This proposal seeks to enable development of therapeutics through synthetic biology methods, in which the molecule's biosynthetic pathway is engineered in order to alter the product. The Class D flavin monooxygenases are found in numerous natural product biosynthetic pathways, including those of valanimycin and daunorubicin, two medicinally useful natural products. With this research we hope to make the Class D flavin monooxygenases of these representative biosynthetic pathways amenable to engineering for synthetic biology purposes. The enzyme-catalyzed step of interest here is a biosynthetic step common to multiple natural products ? flavin-dependent hydroxylation of a primary amine. The enzymes responsible for this step in the two biosynthetic pathways ? vlmH and DnmZ, respectively ? will be biochemically characterized using transient- state kinetics. Site-directed mutagenesis of active site residues will be combined with enzymatic activity and binding studies to validate mechanistic steps and substrate binding interactions. The effect of changes in the substrate binding site on the kinetics of intermediate formation will be investigated for use in validating modifications to the enzyme's substrate specificity. The data yielded will enable rational design of vlmH and DnmZ to alter their substrate binding preferences. Similar studies can be applied to other enzymes of the pathways to introduce diversity into the molecules' final structures.